Histological Specimen Treatment

ABSTRACT

A single dissolving compound forms plural azeotropes, which can be azeotropically vaporized off at various stages of the treatment process, thus maintaining predictable concentrations of the chemicals present. The treatment process can be performed in the absence of formalin or related compounds which can interfere with the preservation of genetic material. A process for preserving a specimen includes using a dissolving compound that can form a plural number of azeotropes, at least one azeotrope being formed between one or more components of the dissolving compound and specimen-supplied water, and at least one azeotrope being formed between different components of the dissolving compound; successively and azeotropically vaporizing off formed azeotropes; and impregnating the specimen with a support medium.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional (and claims the benefit of priorityunder 35 USC 121) of U.S. application Ser. No. 14/706,389, filed May 7,2015, titled “Histological Specimen Treatment,” which is incorporated byreference.

TECHNICAL FIELD

This invention relates to methods and instrumentation for thepreservation of specimens for histological analysis.

BACKGROUND

U.S. Publication No. 2009/0298172 to Wheeler, hereby incorporated byreference in its entirety, describes a histoprocessing technique.Referring to prior art FIG. 1, the histoprocessing instrumentation 25described in U.S. Publication No. 2009/0298172 includes a hermeticallysealable, pressure and temperature regulated reaction container 11 sizedto hold a number of tissue samples 26. The reaction container isconnected by a network of conduits and valves to a source of meltedParaffin constituted by a Paraffin makeup vessel 27, to a source ofdissolving compound in the form of a solvent regenerator 28 drawing froma chemical tank 29, to a solvent pump 30, to a vacuum pump 31, and to anoverflow reservoir 32. The solvent pump is designed to operate over awide range of temperatures and pressures.

As shown in prior art FIG. 2, the solvent regenerator 28 of U.S.Publication No. 2009/0298172 is connected to a solvent distillationassembly 33 including an accumulator 34, a recirculation pump 35 and acondenser 36. A thermocatalytic oxidizer 37 is used to breakdown wastegases into water and carbon dioxide. A carbon bed device could besubstituted for the oxidizer. The waste material extracted from the useddissolving compound is sent to a waste tank 38. A process heater 39(FIG. 1) is provided between the solvent pump 30 and the reaction vessel11. A heat exchanger 60 consisting of a 500 watt, explosion-proof bulbis mounted inside the solvent regenerator 28. A jacket heater 40surrounds the solvent regenerator 28. A similar jacket heater 65surrounds the reaction container 11. A series of snubber protectedpressure sensors 64, 72 and 73 are connected to various areas of thesystem to regulate pressures.

Valves 41-59, and the pumps 30, 31 and 35 are controlled by aprogrammable electronic unit 41 according to techniques well known inthe industrial process arts.

U. S. Publication No. 2009/0298172 describes a process of treatingtissue specimens. Tissue specimens are loaded into a stainless steel orpolymer constructed basket, and then placed into the reaction container11. The container cover is then closed and latched. Proximity sensorsdetect the lid and the process starts. Valve 49 opens and the vacuumpump 31 is started. The pressure of the system is reduced below ambientatmospheric pressure. Valve 49 closes, the vacuum pump is turned off andthe system comes to pressure equilibrium. If this condition is notachieved within one minute, a leak is assumed, and the system is shutoff.

Preconditioned dissolving compound is transferred from the solventregenerator 28 to the reaction container 11. Valves 43, 44 and 45 areopened and the reactor jacket heater 65 is energized. The solvent pump30 and heater 39 are energized. Hot compound fills the reactioncontainer until a solvent level detector switch 67 is triggered. Thepressure in the reaction container is regulated by opening valve 47 andthe reactor's pressure sensor 64. Valve 45 closes and valve 42 opens.The dissolving compound is recycled and heated in the closed loopconsisting of valves 42, 43, and 44, heater 39, solvent pump 30, and thereaction container 11. At this time, the solvent pump 30 is used to pumpup the reactor 11 to an operating pressure as high as about 3.3 bars (50psig).

Specimens are exposed to the dissolving compound for 10 to 30 minutes.During this exposure, cellular solutes are extracted, (e.g., water,lipids, etc.), and replaced with a mixture of liquid Paraffin and thelow molecular hydrocarbons of the compound. Valve 42, 43 and 44, theprocess heater 39, and the solvent pump 30 are turned off. Hotdissolving compound is transferred from the reaction container 11 to thesolvent regenerator 28. The transfer path includes valves 42, 43, and44. Valve 47 is opened between the reaction container and the solventregenerator in order to equilibrate pressure. The transfer lasts untilthe container's low level indicator switch 63 is reset. At this time,valve 42 is closed and the compound is recycled and reconditioned aslater explained. The reaction container 11 is next flooded with liquidParaffin as follows. Valves 42 and 49 open, the vacuum pump 31 is turnedon. Liquid Paraffin flows into the container until the level indicatorswitch 67 is triggered. A high level indicator switch 68 in thecontainer acts as a fail-safe device. At this time valve 42 is closed.The saturated specimens are then subjected to a vacuum to extractvolatiles.

The pressure in the container is further reduced to evaporate alldissolving compound present. The diffusion pump 66 is used to reducepressures to less than 1 torr. Vacuum is applied until pressureequilibrium is achieved, e.g. about 0.9 to 0.99 At. (−27 to −29.91inches Hg), depending upon solvent. Once equilibrium is reached, allvolatile solvent molecules have been removed from the reaction containerand specimens. The process is allowed to continue for an additional 10to 30 minutes depending upon total mass of specimens.

The vacuum system consists of the vacuum pump 31, the diffusion pump 66,and the overflow vessel 32 equipped with a proximity sensor 71, and anisolation valve 49. The overflow vessel acts as an additional fail-safedevice in case of failure of the high level indicator switch 68. The airmakeup valve 50 is provided to dilute gases prior to entering thethermocatalytic oxidizer 37 and to supply cooling gases to the reactioncontainer during cool down cycles. The pressure sensors 64, 72 and 73are connected to the control unit 41 in order to monitor all processesof the instrument. Snubbers are employed to prevent liquid from enteringthe pressure sensors.

In a final step, the Paraffin is returned to the Paraffin makeup vessel27 as follows. Valves 42, 49 and 50 are opened and the vacuum pump 31 isturned off. Cooling air is drawn through valve 50 and routed to thereaction container 11. Paraffin is gravitated to the Paraffin makeupvessel 27. During this time, the specimen temperature drops below theParaffin melt point. The lid of the container opens, the specimen trayis withdrawn and the specimens are extracted and separated.

U.S. Publication No. 2009/0298172 describes that the following alternatebatch solvent blending process may be practiced to prepare aParaffin-loaded dissolving compound.

Referring to prior art FIG. 2, the reaction container 11 is hermeticallysealed. A measured volume of Paraffin-free based solvent is drawn fromthe chemical tank 29 through a coupler 75 and transferred to the solventregenerator 28 where it is heated to 60° C. The jacket heater 40 of thesolvent regenerator and the vacuum pump 31 are energized. A normallyclosed control valve 42 is opened and liquid Paraffin is transferredfrom the Paraffin makeup vessel 27 to the reaction container 11 until aParaffin level detector 62 inside the reaction container is triggered.At this time, the control valve 42 is closed, and the vacuum pump 31 isstopped. Control valves 43, 44, and 45 are opened and solvent istransferred from the solvent regenerator 28 to the reaction container11. When the level detection switch 67 in the reaction container istriggered, the solvent pump 30 is stopped. At this point, all controlvalves are positioned to create the loop configuration describedearlier. The process heater 39 and the solvent pump 30 are energized.The solvent and Paraffin are allowed to blend for about ten minutes intothe final dissolving compound. The circuit is reconfigured to transferthe entire blended compound to the solvent regenerator 28. The reactioncontainer 11 is evacuated. The system is now ready to process specimens.

A process for solvent recovery and regeneration is also described inU.S. Publication No. 2009/0298172 and illustrated in prior art FIG. 2.The objective is to recover, purify, and re-use extraction solvents byisolating and filtering cellular solutes from the dissolving compoundfor waste disposal. U.S. Publication No. 2009/0298172 also describes atechnique for dissolving compound waste gas disposal.

U.S. Publication No. 2009/0298172 describes temperature and pressurepreconditioning of the dissolving compound as follows.

The cellular solute extracting mixture is loaded into the chemical tank29. A bottle coupler 75 provides a connection for solvent transfer andventing. Valves 46, 44 and 43, are open and the solvent pump 30transfers mixture into the solvent regenerator 28. Valves 47 and 49 areopened to provide a path to the thermocatalytic oxidizer 37. Once themixture is transferred from the chemical tank to the solventregenerator, valves 46 and 47 close. The transfer is monitored by levelcontrol switches. Valve 45 is opened and the mixture is heated in theregenerator loop, using the process heater 39 and the regenerator jacketheater 40.

Vent gases are delivered to the thermocatalytic oxidizer 37 foroxidation to carbon dioxide and water as follows. Valve 50 is opened,the vacuum pump 31 is energized. Waste gases are delivered to theoxidizer. Once the inline thermal conductivity detector detects roomconditions, valve 49 and 50 close and the vacuum pump is turned off. Themixture is heated to the desired temperature and pressure, e.g. 60° C.and about 0.8 bars (12 psig). It should be noted that the solventregenerator operating conditions are flexible, and that it can heatedfrom 20 to 100° C. and pressurized to from 1 to about 3.5 At. Inaddition, the variable flow and pressure solvent pump will operateequally over a wide range of temperatures and pressures. A flow orificemay be placed on the discharge to insure proper pump lubrication. Also,it is worth noting that the Paraffin present in the solvent alsoprovides lubricating properties for pumps and valves.

SUMMARY

A single dissolving compound forms plural azeotropes, which can beazeotropically vaporized off at various stages of the treatment process,thus maintaining predictable concentrations of the chemicals present.The treatment process can be performed in the absence of formalin orrelated compounds which can interfere with the preservation of geneticmaterial.

According to one aspect, a process for preserving a specimen includesusing a dissolving compound that can be converted to a gas withoutdamaging the specimen. The dissolving compound that extractsspecimens-supplied water can be azeotropically converted to a gas at aplural of temperatures and pressures. The dissolving compound can beremoved from the specimen by converting dissolving compound componentsto a gas by component vaporization and/or by forming one or moreazeotropes at a plural of temperature and pressures. The dissolvingcompound can be converted to a gas and impregnates the specimen withsupport medium.

According to another aspect, a process for preserving a specimenincludes using a dissolving compound that can form a plural number ofazeotropes, at least one azeotrope being formed between one or morecomponents of the dissolving compound and specimen-supplied water, andat least one azeotrope being formed between different components of thedissolving compound; successively and azeotropically vaporizing offformed azeotropes; and impregnating the specimen with a support medium.

Embodiments of this and other aspects may include one or more of thefollowing.

The successively and azeotropically vaporizing off the azeotropes occursat successively increasing boiling point temperatures. The azeotropesform at successively higher temperatures.

The dissolving compound comprises methanol, tetahydrofuran, toluene, andzinc acetate. The azeotropes formed comprise tetrahydrofuran-methanol,toluene-methanol, tetrahydrofuran-water, and toluene-water.

According to another aspect, a process for preserving a specimenincludes dissolving and removing cellular solutes in the specimen usinga dissolving compound; partially removing the dissolving compound byforming two or more azeotropes with component agents of the dissolvingcompound, and azeotropically vaporizing off the two or more azeotropes;and replacing the solutes with an impregnated support medium.

Embodiments of this and other aspects may include one or more of thefollowing.

The process is performed in a continuous sequence of steps within asingle vessel. The process is performed in the absence of formalin. Theimpregnated support medium includes paraffin. The impregnated tissuesupport medium is selected from paraffin, plastic polymers, polyvinylalcohol, polyethylene glycols, waxes, cellulose derivatives, agars,gelations, and sugars.

According to another aspect, a process for preserving a specimenincludes removing from the specimen cellular solutes that interfere withpreservation and replacing the removed solutes with an impregnatedsupport medium. The removing includes extracting an amount of watersupplied by the specimen. The extracting includes dissolving the solutesincluding the amount of water using a dissolving compound; forming anazeotropic water-containing mixture of the amount of water and acomponent of the dissolving compound; azteotropically vaporizing off anamount of the azeotropic water-containing mixture. The process includesforming a second azeotropic mixture of at least two components of thedissolving compound; and azteotropically vaporizing off an amount of thesecond azeotropic mixture.

Embodiments of this and other aspects may include one or more of thefollowing.

The removing includes clearing an amount of the dissolving compound fromthe specimen. The clearing includes forming an azeotropicfixitive-containing mixture of an amount of a fixitive agent componentpresent in the dissolving compound and another component in thedissolving compound; and azteotropically vaporizing off an amount of theazeotropic fixitive-containing mixture.

The removing includes clearing an amount of the dissolving compound fromthe tissue. The clearing includes forming an azeotropicdehydrant-containing mixture of an amount of a dehydrant agent componentpresent in the dissolving compound and another component in thedissolving compound; and azteotropically vaporizing off an amount of theazeotropic dehydrant-containing mixture.

The impregnated support medium includes paraffin. The impregnatedsupport medium is selected from paraffin, plastic polymers, polyvinylalcohol, polyethylene glycols, waxes, cellulose derivatives, agars,gelations, and sugars.

The dissolving compound includes a single component or groups ofcomponents that is removed by vaporization from the specimen withoutdamage to the specimen. The dissolving compound includes a singlecomponent or groups of components that release formaldehyde from thespecimen and convert it to a monomeric gas. The dissolving compoundincludes a support medium component.

The dissolving compound includes a fixative component, a dehydrantcomponent, and a clearing agent component. The dissolving compoundincludes a cation as a cellular anti-rupturing agent. The cationcellular anti-rupturing agent includes a compound selected from thegroup of zinc acetate, iron acetate, magnesium acetate, zinc citrate,iron citrate, and zinc malate. The fixative component is selected fromthe group of formalin, glacial acetic acid, zinc chloride or zincsulfate, chromic acid, ethanol, methanol, acetone, mercuric chloride,picric acid, and potassium dichromate. The dehydrant component isselected from the group of alcohols, isopropyl alcohol, ethanol,denatured alcohols, butanols, ethers, ketones, dioxane, and acetone. Theclearing agent component is selected from the group of xylene, toluene,benzene, acetone, butanols, dioxane, methyl salicylate, cedarwood oil,hexane, heptane, octane, and decane. The dielectric constant of theclearing agent component is between 2.0 and 10. The dissolving compoundincludes a mixture of between about 0 and 30% by weight alcohol, betweenabout 45 and 99% by weight ether, and between about 0 and 20% by weighthydrocarbon.

The process includes prefixing the specimen using an amount of fixativeincluding a mixture of between about 20 and 50% by weight methanol, andbetween about 20 and 50% by weight ethanol. The mixture is saturatedwith anhydrous ammonia. The mixture has a pH maintained between about5.0 and 5.4 through the application of an effective amount of glacialacetic acid. The mixture includes an amount of vanadyl-ribonucleosidecomplex or RNAlater® as a nucleic acid preservative.

The process includes prefixing the specimen using an amount of fixativeincluding a mixture of between about 80 and 98% by weight methanol,between about 1 and 20% by weight acetonitrile, and between about 1 and5% by weight EDTA. The mixture has a pH maintained between about 5.0 and5.4 through the application of an effective amount of glacial aceticacid.

According to another aspect, a dissolving compound includes one or morepre-polymers that polymerize to form a support medium. The dissolvingcompound is convertable to gas without damaging one or more specimens ata plurality of temperatures and pressure.

Embodiments of this and other aspect may include one or more of thefollowing.

The dissolving compound includes volatile components that areconvertible to gas at a pressure greater than 10 torr and a temperatureof no more than about 150° C. The support medium is UV curable. Thedissolving compound is a pre-fixative. The dissolving compound containsone or more photo-initiators. The dissolving compound contains2-hydroxyethyl methacrylate and triethylene glycol dimethacrylate. Thedissolving compound contains urethane acrylate, ethoxylatedtrimethylolpropane triacrylate, methacrylic acid and 2-hyrdoxyethylmethacrylate. The dissolving compound contains one or more plasticizer,for example, phthalates, benzoates, adipates or trimelliates.

The details of one or more embodiments of the invention are set forth inthe accompa-nying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a prior art diagrammatical schematic illustration of anapparatus for preserving a specimen.

FIG. 2 is a diagrammatical schematic illustration of the solventregeneration portion of the apparatus.

FIG. 3 is a flow diagram of tissue histology according to an exemplaryembodiment of the invention.

FIG. 4 is a flow diagram of a tissue preserving process according to anexemplary embodiment of the invention.

FIG. 5 shows an H&E stained Uterus specimen processed using theazeotropically removed dissolving compound of Example 3.

FIG. 6 is a CytoScan brand analysis output for a uterus specimen afterprocessing employing the azeotropically removed dissolving compound ofExample 3.

FIG. 7 shows an H&E Lung Adreno specimen processed using theazeotropically removed dissolving compound of Example 4.

FIG. 8 shows a specimen process using azeotropically removed dissolvingcompound of Example 5.

FIGS. 9A and 9B are graphs of azeotropic behavior of a ternary mixtureused in specimen preservation.

DETAILED DESCRIPTION

Referring to the drawings there is shown in FIG. 3 a flow diagram oftissue histology 1. First a biopsy 2 is performed to obtain the tissuespecimen to be preserved and analyzed. The fresh tissue specimen 3 canbe sent directly to preservation processing 10. Alternately, the freshspecimen can undergo a pre-fixation process 5 before being sent topreservation processing. The histology process can also be performed onplant or vegetable matter.

After preservation 10, some or all of the preserved tissue specimen canundergo standard “classical” pathological analyses 6, and/or some or allof the preserved tissue specimen can undergo a DNA/RNA extractionprocess 7, where the extracted genetic material can be supplied to oneor more genetic analysis processes including downstream enzymaticreaction analysis 9 a, cytogenetic analysis using microarray technology9 b, and/or next generation sequencing 9 c or molecular analysis.

In the prior art preservation process described above, two azeotropesare formed. One of the azeotropes, acetone-hexane, is formed with thedissolving compound. The other azeotrope (hexane-water) is not formedwith the dissolving compound. In preservation process 10 two or moreazeotropes are formed with the dissolving compound.

Referring to FIG. 4, the tissue specimen preserving process 10 includes,in a hermetically sealable, and pressure and temperature controllablereaction vessel or container 11 in which one or more tissue specimensare being held, admitting at 12 a limited volume, for example, about 200ml of melted tissue support medium such as paraffin from a storage tankat 13. The paraffin has been preferably conditioned at 14 by raising itstemperature slightly above its melting point, for example, 60° C., suchthat heated support medium in a liquid state is admitted into thecontainer 11. The limited paraffin admission at 12 mixes withsubsequently introduced dissolving compound and acts to decrease theinfiltration time into the specimens of further subsequently introducedparaffin.

A dissolving compound formulated to dissolve cellular solutes in thespecimens is then introduced at 15 into the container 11. These cellularnon-volatile solutes that are dissolved by the dissolving compoundinclude lipids: glycerides, triacylglycerols, phospholipids,sphingolipids, fatty acids and cholesterol. Other extractible speciesinclude soluble vitamins, cellular waste products and a host of otherbiochemical molecules. There are two types of cellular water present inthe specimens, bound and free water. Only free water is extracted by thedissolving compound. The components of the dissolving compound now mixedwith the paraffin introduced at 12 penetrate the specimens at 16, withthe dissolving compound diluting and extracting solutes while thespecimen becomes saturated with the dissolving compound.

The solutes that are diluted and extracted from the specimens as well asexcess dissolving compound that did not enter the specimens areevacuated from the container 11 in liquid form at 17 and returned to theregenerator at 21. Formaldehyde that may be present in the specimen fromfixation is converted by the dissolving compound from bound formaldehydeto a monomeric gas free formaldehyde. During an evaporation cycle at 17,the monomeric gas trapped in the top region of the container 11 ispulled out through a vent at the top of the container 11 at 22.

To remove the residual dissolving compound present in the tissue, thepressure and temperature in the container 11 are controlled at 17 tovaporize the residual dissolving compound and form a plurality ofazeotropes of the dissolving compound, as described further below. Inparticular, a vacuum is pulled to remove dissolving compound from thespecimen through component evaporation and azeotrope formation. Thetemperature and pressure are controlled in an evaporation cycle to forma distinct azeotrope molecular species of cellular free water withcomponents of the dissolving compound. Further azeotropes are alsoformed from components of the dissolving compound. All of the azeotropegaseous species and the free formaldehyde are removed from container 11through the vent at 22.

The paraffin from the dissolving compound/paraffin mixture that had beendeposited in the specimen from the dissolving compound saturation of thespecimen remains in the specimen. The container 11 is then flooded at 18with about 5 liters of paraffin mixed with dissolving compound, whichsaturates the specimens. The dissolving compound mixed with the paraffinis an entrained amount absorbed by the paraffin, and the dissolvingcompound acts to decrease the viscosity and the infiltration time of theparaffin. The pressure in the container 11 is then reduced and thetemperature controlled such that components of the dissolving compoundare vaporized and azeotropes are produced at 18 in the same manner asdescribed at 17. The gaseous species are again removed through the ventat 22. After removal at 19 of excess paraffin in liquid form from thecontainer 11, the container is cooled at 20 allowing retrieval of thetreated specimens.

The evacuated dissolving compound components carrying the removedsolutes can be regenerated by distillation at 21 and by converting at 22the waste gases into carbon dioxide and water through a thermocatalyticoxidizer. The recovered dissolving compound can then be stored at 23 andthen pressure and temperature preconditioned at 24 prior to being usedagain in the container 11 such that heated and pressurized dissolvingcompound is introduced into the container 11 at 15.

In steps 17 and 18, the pressure and temperature in the container isadjusted to successively azeotropically evaporate the dissolvingcompound components present in the specimens. For example, FIGS. 9A and9B demonstrate the complex azeotropic behavior of a ternary dissolvingcompound mixture of methanol, acetone, and chloroform used in specimenpreservation. The mixture forms three binary azeotropes and one ternaryazeotrope at atmospheric pressure. A binary azeotrope of chloroform andmethanol has a boiling point of 53.6° C.; a binary azeotrope of acetoneand methanol has a boiling point of 55.5° C.; a binary azeotrope ofacetone and chloroform has a boiling point of 65.5° C.; and a ternaryazeotrope of methanol, acetone, and chloroform has a boiling point of57.6° C. A general property of azeotropes is that a variation inpressure changes the boiling temperature and the composition of themixture. The methanol and acetone act as the fixative, the acetone actsas the dehydrant, and the acetone and chloroform act as the clearingagent.

The three binary azeotropes and one ternary azeotrope together with thethree pure components form a system having seven nodes which altogetherform four distallation regions. Two nodes are stable (pure methanol andthe binary azeotrope of chloroform and acetone), which both have thelowest vapor pressure (isothermal calculation) in the two regions theyare part of. The other two binary azeotropes are unstable nodes. Theyhave the highest vapor pressure in their regions. The other nodes aresaddles (the ternary azeotrope, the pure acetone and the purechloroform). The border lines in this system connect the ternaryazeotrope (saddle) with the two stable nodes and the two unstable nodes.The residue curves are always moving away from an unstable node to asaddle but never reach a saddle because the residue curves then turn toa stable node.

As discussed further below, successive azeotropes are formed, forexample, as the temperature is raised to 60.7° C., 63.5° C., and 64° C.Each azeotrope is vaporized off by using the diffusion pump 66 to reducethe pressure. Vacuum is applied until pressure equilibrium is achieved,for example, about 0.9 atmospheres to about 0.99 atmospheres (−27 to−29.91 inches Hg), depending upon the component being removed. Onceequilibrium is reached, all volatile solvent molecules have been removedfrom the reaction container and specimens. After the first azeotrope isvaporized off, the temperature is raised to form the next azeotrope, andso on. The process is allowed to continue for each additional componentsfor up to 10 to 30 minutes depending upon the total mass of specimens.The rise in temperature and the evacuation of gasses through the vent at22 occur at the same time.

Dissolving Compound

The dissolving compound can be selected from various mixtures ofcomponent chemicals for use in the above described tissue preservationprocess and associated apparatus. The chemicals or mixtures thereof canact as one or more functional components of the dissolving compound.These functional components include a fixation agent component, adehydration agent component, and a clearing agent component. Optionally,the dissolving compound can be blended with an impregnation agent suchas paraffin. Optionally, dissolving compound can also include a cation(examples include but are not limited to zinc acetate, iron acetate,magnesium acetate, zinc citrate, iron citrate, and zinc malate) as acellular anti-rupturing agent.

Significantly, the dissolving compound is a mixture of component agentsthat form one or more azeotropes during different stages in the process,and thus the dissolving compound and its components can be removedwithout damaging the specimen. Azeotropes can be used to extractcellular water from the tissue, remove components of the dissolvingcompound from the tissue, and deposit dissolved paraffin or other mediainto the tissue specimen. Therefore, each step in the tissue processing,namely fixation, dehydration, clearing and infiltration, can beaccomplished using the supplied dissolving compound. In this way, asingle dissolving compound can be used to perform all the stepsnecessary for tissue preservation.

Further, it shall be understood that, if desired, the dissolvingcompound can be selected to perform a fewer number of the preservativefunctions. So, for example, a dissolving compound can be selected tofix, dehydrate and clear only, or selected to dehydrate and clear only.Accomplishing the process in whole or in part depends upon the chemicalsemployed and specimen type.

Further, the dissolving compound can contain one or more components thatform one or more azeotropes in the presence of paraffin or other supportmedia used in the infiltration step. Support media other than paraffincan include but are not limited to polymeric compounds, likepolyethylene glycols, waxes such as carbowax, resinous or acrylicspolymers, cellulose derivatives, agars, gelations, and sugars. Thesupport media can be composed of monomeric, cross linking agents and/orcopolymers that infiltrated the specimen and cure, to produce apolymeric support medium. These compounds are added to the prefixativereagent and/or the dissolving compound and may contain photoinitiatorsto accelerate the polymerization reaction. Examples of pre-polymercompounds include, but are not limited to, Urethane acrylate,methacrylic acid, 2-hydroxyethly methacrylate, ethoxylatedtrimethylolpropane triacetate, triethylene glycol dimethacrylate, andhexadecyl methacrylate. The paraffin or other support media can varybetween 0 and 20 percent by weight of the combination of dissolvingcompound and support media.

In general, common chemical compounds that can be used to form theazeotropically removed dissolving compound used in tissue preservationinclude but are not limited to alcohols, organic acids, alky halides,esters, ketones, aldehydes, ethers, aromatics, solvents, oxygenates andhydrocarbon blends.

During the fixation stages, the removal of dissolving compoundcomponents is not yet required. In other words, fixation can occur priorto the removal of any of the components of the dissolving compound. Inaddition fixation can also occur during removal of the dissolvingcompound, where the fixative is vaporized with one or more components ofthe dissolving compound.

During dehydration stages, water and hydrated solutes can be extractedfrom tissue using liquid-liquid extraction using more than one reagentof similar chemical composition. Components of the dissolving compoundform hydration spheres with free water, creating a compound that can bevaporized at temperatures that will not damage the tissue specimen.Thus, when using a single reagent, the final amount of water andresidual dissolving compound is removed by azeotrope formation. Intissue preservation processing, azeotropes used to remove water fromtissue and the dissolving compound are those that have an azeotropicboiling point of 150° C. or less, more preferably 115° C. or less, andmost preferably less than 100° C. so that the tissue is not damaged bythe temperature of boiling.

Components that form azeotropes with water include, but are not limitedto ethanol, n-propanol, isopropanol, n-butanol, sec-butanol,iso-butanol, tert-butanol, ally alcohols, formic acid, propionic acid,butyric acid, iso-butyric acid, ethylene chloride, propylene chloride,chloroform, carbon tetrachloride, methyl chloride, ethyl acetate, methylacetate, n-propyl acetate, ethyl nitrate, methyl ethyl ketone, pyridine,benzene, toluene, cyclohexane, hexane, heptane, octane, m-xylene,p-xylene, ethylbenezene, diethyl etther, tetrahydrofuran, anisole,acetonitrile, chloral, and hydrazine.

Azeotropes can also be used during clearing stages to remove componentsof the dissolving compound, such as the fixative, the dehydrants, andclearing agents themselves or any combination thereof. The dissolvingcompound can be composed of one or more azeotropes containing a clearingagent. Examples of clearing agents include but are not limited to methylsalicylate, cedar wood oil xylene, toluene, benzene, aromatic solvent150, terpineol, carbon tetrachloride, chloroform, dioxane, celloslove,tetrahydrofuran, and butanols.

Finally, the dissolving compound is designed to be vaporized in a mannerthat results in the preservation of tissue specimens without damage tothe specimen, and the dissolving compound is used to deposit supportmedium to the tissue specimen through vaporization.

During tissue processing a small amount of dissolving compound istrapped in the lines from vessel 11 and valve 42, and is transferredinto the paraffin storage at 13. The dissolving compound is designed tobe entrained within the paraffin. This modifies the paraffin so that itsviscosity is reduced for faster infiltration. The dissolving compoundpresent in the paraffin storage vessel is regulated so that the amountof entrained dissolving compound is approximately constant. Thus, duringparaffin infiltration and storage, water present in the paraffin can beremoved from the paraffin by azeotrope formation at 13.

Prefixing

Fresh tissue can be prefixed, that is, undergo a fixation process priorto being processed by the dissolving compound. For example, the biopsiedtissue can be first exposed to a solution of ten to twenty percentformalin. Exposure time may vary from 2 minutes to 48 hours. After thisexposure the tissue is processed in a tissue processor using adissolving compound which forms one or more azeotropes, as describedabove.

Prefixation can also be done using a fixative which is intended topreserve genetic material and avoids the use of formalin. For example, asolution of 20 to 50 percent by weight ethanol and 20 to 50 percent byweight methanol is bubbled with anhydrous ammonia until saturated. ThepH of this saturated solution is maintained between 5.4 and 5.0 usingglacial acetic acid. Vanadyl-ribonucleoside complex or RNAlater brandreagent commercially available from Qiagen of Hilden, Germany can beadded to further preserve the nucleic acids. The tissue specimens can betreated for up to 12 hours, to preserve RNA. Thereafter, the prefixedtissue can be processed using the dissolving compound, as describedabove. The processed tissue specimens can be stored for 5 years, withoutderogation of its DNA. These tissue specimens may be extracted andsequenced or analyzed for nucleic acids using microarray-based testing(cytogenic array) as well as conventional pathology testing.

In an alternate example of prefixation in order to preserve geneticmaterial a solution of methanol 70-98 percent by weight and acetonitrile1-20 percent by weight, and EDTA 1-5 percent by weight are blended, andglacial acetic acid is added to maintain a pH between 5.4 and 5.0. Thetissue specimens may be treated for no more than 24 hours to preserveRNA, and then processed using an azeotropically removed dissolvingcompound tissue process, as described above. The processed tissuespecimens can be stored for up to 5 years and analyzed for nucleic acidsusing microarray-based clinical testing as well as traditional pathologytesting.

Specimens Protocol for Processing Using Dissolving Compound Protocol 1:Specimen Prefixing Formaldehyde Free or Formalin Fixed Specimens

-   -   Ensure that the grossed sections are thin, preferably no more        than 3 mm thick.    -   Place specimen in fixative as soon as possible after the blood        supply has been interrupted.    -   Open specimens wherever possible. Gastrointestinal specimens        should be opened, pinned to a cork or paraffin wax board and        placed in fixative. Uterus specimens should also be opened and        placed in fixative. Lungs can be inflated with fixative by        gravity flow.    -   Slice specimens, such as spleen, breast, kidney, any organ        resection, or large tumor into slices and place in fixative.    -   Bisect lymph nodes when appropriate and place in fixative.    -   Place fixative container for holding cassettes on stir plates,        and provide agitation to enhance the fixation and penetration        process.    -   Use only fresh fixative solution. Ensure that the fixative        volume is 15 to 20 times the tissue volume.    -   Well-grossed sections of routine tissue should be fixed at least        8 to 12 hours to ensure at least adequate fixation, but no more        than 24 hours.

For processing of fresh tissue specimens, where the specimens meet thedefinition of needle biopsies (up to 1 mm thick), the protocol forPrefixing Formaldehyde Free above is followed for transport to thelaboratory, omitting the last step of fixing for at least 8 to 12 hours.

Stain Protocol: after the specimens are processed as described abovewith reference to FIG. 4, and embedded in a paraffin block, cut andplaced on a slide, Hematoxylin and Eosin Y are used to stain thespecimens on the slide. Steps 1-22 of the staining process are set outin the table below.

1 Xylene 3 min 2 Xylene 3 min 3 Xylene 3 min 4 100% Alcohol 1 min 5 100%Alcohol 1 min 6 100% Alcohol 1 min 7 80% Alcohol 30 sec 8 Wash in H2O 1min 9 Hematoxylin 2 min 10 Wash in H2O 1 min 11 Differentiate 30 sec 12Wash in H2O 1 min 13 Bluing agent 30 sec 14 Wash in H2O 1 min 15 80%Alcohol 16 Eosin Y - Alcoholic 20 sec 17 100% Alcohol 1 min 18 100%Alcohol 1 min 19 100% Alcohol 1 min 20 Xylene 1 min 21 Xylene 1 min 22Xylene 1 min

Example 1

A dissolving compound is selected containing an alcohol, ether, and anaromatic hydrocarbon. The concentration of the components may vary, buttypically the alcohol is between 0 and 30 percent by weight, the etherbetween 45 and 99 percent by weight and the aromatic between 0 and 20percent by weight. The alcohol can act as a fixative component. Theether can act as a dehydrant and clearing component. The aromatic canact as a fixative, dehydrant and clearing component.

The tissue can be exposed to the dissolving compound for a specifiedtime (10-120 minutes) under a specific pressure (50-350 kPa) andtemperature (25-99° C.) in order to completely saturate the specimenwith dissolving compound, and allow liquid extraction of the cellularsolutes. Liquid dissolving compound containing cellular solutes isdrained from the reactor. Thereafter, using temperature and pressurevariations, the remaining dissolving compound is removed from the tissueby the successive formation and vaporization of azeotropes in a properorder. Thereafter, the tissue is suitable for pathological andcytogenetic examination.

Using the alcohol/ether/aromatic dissolving compound, the firstazeotrope that forms is fixative based and is removed by formation of anether-alcohol azeotrope. Thus, at this point, the dehydrant and/orclearing agent can be used to remove the balance of the fixative byvaporizing the developing azeotrope.

Next, water remaining in the tissue reacts with the remaining etherdehydrant to form an ether-water azeotrope which can be removed byazeotropic vaporization. At this stage, by removing most of the water,the tissue's polarity has changed.

Next, the clearing agent can be removed by azeotrope formation and itsazeotropic vaporization.

The rate and amount of removal of these azeotropes is controlled byvarying the pressure and/or temperature of the reactor. Specifically,the pressure varies from 350 kPa to 25 kPa, more specifically between175 kPa and 60 kPa and even more specifically between 100 kPa and 45kPa. Specifically, the temperature varies from about 30° C. to about150° C., more specifically the temperature varies from about 30° C. toabout 100° C.

This example shows that in forming the dissolving compound, one or morealcohols can be blended with a low molecular weight oxygenate and/orhydrocarbon that extract cellular solutes that interfere withinfiltration, and removal of the dissolving compound is achieved by theformation of one or more azeotropes.

This example further shows that an azeotrope mixture can be used to fixand/or dehydrate the tissue being preserved.

Further, this example shows that the clearing of the tissue can beaccomplish by a low boiling point component, where the boiling point canbe less than 150° C. Examples of chemicals useful as a low boiling pointclearing agent component include, but are not limited to C4 through C15hydrocarbons, branches chain alkyl alcohols, C4 through C12 oxygenates,chlorinated hydrocarbons, brominated hydrocarbons, and other aromatichydrocarbon solvents having a boiling point of 150° C. or less.

Example 2

A dissolving compound is selected containing 20 percent by weightmethanol and 80 percent by weight tert-butanol. The dissolving compoundis charged to the container 11 containing tissue specimens at 40° C. Thespecimen is exposed to the dissolving compound for 30 minutes and thedissolving compound is drained from the reactor. The reactor temperatureis slowly elevated to 65° C. while the pressure is varied between 50 and200 kPa. The residual methanol is converted to a gas and is ventedduring the pressure cycle. In this example the methanol is removed as azeotrope. After a period of time the reactor temperature is raised to80° C. and the tert-butanol slowly forms an azeotrope with residualwater present in the tissue. During the temperature change and at theisotherm of 80° C., the reactor pressure is varied between 50 and 200kPa. The azeotrope is removed from the reactor using the vent.

A solution containing Carbowax as the support medium is then charged tothe reactor for impregnating the tissue. Pressure variation (forexample, between 50 and 200 kPa) is again used to vaporize the residualtert-butanol/water azeotrope. The reactor temperature is dropped to 68°C., while the pressure variation continues, and the specimens areexposed to the carbowax medium for an additional 30 minutes.

Example 3

A dissolving compound is selected to include the chemicals shown inTable 3.1 below.

TABLE 3.1 Selected dissolving compound Component Relative Weight PercentMethanol 15.2 Tetahydrofuran (THF) 79.0 Toluene 5.4 Zinc acetate 0.4

Tissue specimens of the type and volume shown in Table 3.2 were loadedinto the container 11.

TABLE 3.2 Specimen type and volume Tissue Dimensions (mm) Intestine 15 ×10 × 5 Heart 25 × 15 × 6 Kidney 30 × 25 × 5 Breast 30 × 25 × 6 Liver 20× 15 × 5 Uterus 20 × 25 × 6

Tissue Processing: The dissolving compound described in Table 3.1 isstored at a temperature of 40° C. The heated dissolving compound is thentransferred to the container 11 containing tissue specimens listed inTable 3.2. The reaction container's conditions are next equilibrated to45° C., with a reactor flow rate of 4.0 l/min. Specimens are processedin the dissolving compound for 70 minutes.

During this processing time, zinc acetate is converted to azinc-plasma-membrane chelate compound, and all tissue specimens becomesuper saturated with dissolving compound. The zinc chelate helps provideincreased cellular membrane integrity to limit tissue cell rupturing.The reactor container skin temperature is increased to 60° C., and thedissolving compound is returned to the storage vessel. Liquid paraffinat 59-72° C. is then added to the reactor container. Residual dissolvingcompound within the reactor container blends with the paraffin. Thetemperature of the solvent-paraffin blend is slowly increased to 68° C.and the pressure is varied between 50 and 200 kPa.

As the temperature of the dissolving compound/paraffin blend increases,four azeotropes are formed (Table 3.3). The dissolving compound andwater and their azeotropes are removed from the reactor during thepressure variation and the gas is vented.

TABLE 3.3 Example of azeotropes that form during specimen processing.Table shows relative boiling points corrected to atmospheric pressurefor reference only. Relative Boiling point of Azeotrope AzeotropeAzeotrope at atmosphere Component 1 Component 2 conditions (C. °)Tetrahydrofuran (THF) Methanol 60.7 Toluene Methanol 63.8Tetrahydrofuran Water 65 Toluene Water 84.1

It is important to note that the formation of successive azeotropesallows for the removal of all the dissolving compound components and allthe cellular solutes being removed from the tissue at a temperature thatis below their normal boiling point. For example, water boils at 100° C.without azeotrope formation, and toluene boils at about 110° C. Atemperature of 100° C. would damage and destroy the tissue specimens,rendering them unsuitable for diagnosis. In addition, the presence ofthe selected dissolving compound results in a decrease in paraffinviscosity, thus increasing paraffin penetration, and resulting in idealprocessing for large tissue specimens.

FIG. 5 shows an H&E stained Uterus specimen processed using theazeotropically removed dissolving compound components listed in Table3.1. The results show excellent tissue preservation. Further, in thisway, tissue processing using plural and successive azeotrope formationresults in a faster tissue preservation method when compared toconventional FFPE (Formalin Fixed Paraffin Embedded) methods.

FIG. 6 shows a CytoScan brand analysis of uterus specimen afterprocessing employing an azeotrope dissolving compound listed in Table3.1 using GeneChip Scanner 3000 brand analysis equipment. The CytoScananalysis and GeneChip Scanner 3000 equipment are commercially availablefrom Affymetrix of Santa Clara, Calif.

Example 3b

Specimens whose volume ranges from 1 mm³ to 5000 mm³ are placed in thereactor container 11. The reactor is sealed and a dissolving compoundcontaining 185 grams of methanol, 960 grams of tetrahydrofuran, 250-400ppm of 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 66 grams of toluene and5 grams of zinc acetate are charged into the reactor container atbetween 25° C. and 40° C. The dissolving compound is circulated at avariable rate between 1 and 10 liters per minute and heated to about 45°C. for a period of 70 minutes. The dissolving compound generates aninternal reactor pressure between 130 and 165 kPa. During this time,there is mass transfer of the cellular solutes that interfere withtissue preservation from the tissue to the bulk phase of the dissolvingcompound. As part of the process the tissue is fixed, dehydrated, andcleared by the dissolving compound. Cellular components that causeautolysis, for example enzymes, are deactivated. In addition, there aresubstantial molecular configurational changes that render the component,for example, nucleic acids, DNA, RNAs, proteins, and carbohydrates,inactive. There is a polarity change in the tissue from polar tonon-polar as more of the tissue interferences are removed.

At about 70 minutes liquid dissolving compound is drained from thereactor container. The reactor container walls are then heated from 45to 68° C., and a slight vacuum of between 100 and 45 kPa is applied tothe reactor. This generates the first azeotrope, namely a THF-methanolcompound having a boiling point at about 60° C.

It is important to note that the azeotrope boiling point temperature isgiven at atmospheric conditions, 760 Torr, for reference only. The trueazeotrope boiling point would be less than 60° C. at 60 kPa.

The azeotropic vaporization removal of the THF-methanol azeotropedecreases the methanol concentration within the tissue. The remainingmethanol is removed by the second azeotrope, namely a toluene-methanolcompound having a boiling point at about 63° C. As this processcontinues the polar methanol is removed and the tissue becomes non-polarand saturated with THF and toluene.

At about this point, paraffin containing a small amount of dissolvingcompound is charged to the reactor. The reactor pressure is reduced toabout 60 kPa for about 1 to 3 minutes, and then pressure is appliedbetween 100-210 kPa, for about 90 seconds. Any methanol present in theparaffin-dissolving compound is removing by azeotrope formation. Theviscosity of the paraffin is also reduced by the presence of thedissolving compound, improving infiltration of the paraffin into thetissue.

The next azeotrope to form is THF-water. Any residual free water presentin the tissue is thus removed by azeotropic boiling of the THF-waterazeotrope at about 65° C. Toluene and THF tend to clear or brighten thetissue, freeing it from cloudiness, muddiness, and blemishes, resultingin a more transparent, and non turbid tissue.

The final azeotrope to form is toluene-water at about 68° C. and 60 kPa.Gases formed in the reactor are removed through the vent as the pressureis varied from about 50 to 210 kPa. A mixture of dissolving compound andparaffin floods the reactor, the reactor pressure is varied from about50 kPa to 210 kPa, the dissolving compound paraffin mixture is heatedfrom 50 to about 100° C., and gases formed are vented. The processingtime varies from 30 minutes to about 4 hours and is dependent uponspecimen volume.

At the end of this process specimens can be stored in liquid paraffinfor about 30 seconds to about 100 hours, upon which excess paraffin isremoved from the reactor, and samples are cooled to room temperature,and are suitable for embedding and microtome cutting. This tissue isthen stained using conventional IHC or FISH stains. The remainingparaffin-loaded tissue blocks can then be stored for up to thirty years.

Example 3c

A dissolving compound is selected containing 79.1 percent by weight THF,15.2 percent by weight methanol, 4.9 percent by weight 2-hydroxethylmethacrylate, 0.5 percent by weight triethylene glycol dimethacrylate,and 0.2 percent by weight of jayflex DUP and the dissolving compound ispreheated to 50° C. The dissolving compound is charged to container 11containing specimens and the specimens are exposed to the dissolvingcompound for 15 minutes. During that time the polymer precursor mixtureinfiltrates the specimens. The liquid dissolving compound is transferredfrom container 11 to container 28. The temperature of container 11 iselevated to 60° C. while the pressure is varied between 50 and 210 kPa.Volatile components and residual cellular water are removed fromcontainer 11 as gas through the vent. The pre-polymer infiltratedspecimen is extracted from the container 11 and cured via UV light. Theprocessed specimen is directly cut using a microtome followed bystaining. The results show excellent tissue preservation.

Example 4

A dissolving compound is selected to include the chemicals shown inTable 4.1 below.

TABLE 4.1 Selected dissolving compound Component Relative Weight PercentMethanol 24 Ethanol 14.6 Hexane 61.0 Lubricity additive 0.4

This dissolving compound is selected to extract tissue solutes thatinterfere with polymer or wax infiltration.

TABLE 4.2 Azeotropes formed Azeotrope Azeotrope Azeotrope RelativeBoiling point of Component Component Component Azeotrope at atmosphere 12 3 conditions (C. °) Methanol n-Hexane 50.6 Ethanol n-Hexane Water 56.0Ethanol n-Hexane 58.7 n-Hexane Water 61.1

Specimens whose volume ranges from 1 mm³ to 5000 mm³ are placed in thereactor container 11. The reactor is sealed and a dissolving compoundcontaining 650 grams of methanol, 400 grams of Ethanol, 1690 grams oftoluene and 12 grams of lubricity additive are charged into thecontainer at between 25 and 40° C. The operating parameters are employedas in Example 3. Azeotrope boiling points are given in Table 4.2.

FIG. 7 shows an H&E Lung Adreno specimen processed using theazeotropically removed dissolving compound components listed in Table4.1. The results show excellent tissue preservation.

FIG. 8 illustrates a tissue section at room temperature 25° C. withlubricity additive and no ice cooling or skin refrigerant. The specimenis good for further histopathological procedures.

Example 5

A dissolving compound is selected to include the chemicals shown inTable 5.1 below.

TABLE 5.1 Selected dissolving compound Component Relative Weight PercentMethanol 12 tert-Butanol 88

The operating parameters are employed as in Example 3. The dissolvingcompound is removed by vaporizing the methanol (i.e., zeotrope) and thenthe azeotrope shown in table 5.2. All gases formed in the reactor areremoved using the vent.

TABLE 5.2 Zeotrope and Azeotrope used in tissue processing for example 5Relative Boiling point of Compo- Compo- Compo- Azeotrope at atmospherenent 1 nent 2 nent 3 conditions (C. °) Methanol zeotrope Tert-butanolWater 79.9

Specimens whose volume ranges from 1 mm³ to 5000 mm³ are placed in thereactor container 11. The container is sealed and a dissolving compoundcontaining 200 grams of methanol and 800 grams of tert-butanol ischarged into the reactor container at between 30 and 40° C. Theinfiltrating medium is 9 parts Carbowax 4000 and 1 part Carbowax 1500.The operating parameters are employed as in Example 3. Azeotrope boilingpoints are given in Table 5.2.

Ketone/Hydrocarbon Dissolving Compounds

The dissolving compound can be selected to be a mixture of a ketone anda low molecular weight hydrocarbon which results in a simple three stepprocedure performed in a single container 11. First, the specimens aresaturated with a mixture of a ketone/hydrocarbon dissolving compound,e.g., an acetone/hexane (“A/H”) or an acetone/xylene (“A/X”) mixture, todissolve lipids and other cellular solutes. The container is thenflooded with melted paraffin. In a last step, the dissolving compoundmixture is removed from the container by draining and vaporization,allowing the melted paraffin to impregnate the specimens. Raw, i.e.,non-processed and non-burred specimens up to 5 mm thick can be processedin about 60 minutes. A dissolving compound regenerator distills theevacuated dissolving compound, and converts vent waste gases into carbondioxide and water through a thermocatalytic oxidizer.

The ketone/hydrocarbon dissolving compound embodiments may include abuffer to stabilize the extraction solvents to near neutral conditions.Tris-(hydroxymethyl) aminomethane is an excellent buffer for thisapplication, but any compound capable of buffering the solvents at nearneutral pH and which is soluble in the dissolving compound isapplicable.

Other chemicals may be added to the A/H or A/X dissolving compound toincrease viscosity. Examples include alcohols, ketones, aldehydes,solvents, and hydrocarbons, e.g. DMSO and 2-propanol. DMSO is anadditional extraction solvent that can added to the A/H or A/Xdissolving compound. DMSO assists in the extraction of large ringcompounds and free nucleotides, e.g., lithocholic acid, deoxycholicacid, cholesterol, and other compounds that interfere with paraffinimpregnation. Alcohols maybe added to slow the extraction process andprevent cellular membranes from rupturing.

Example 6

In this example, no Paraffin is directly added to the solvent mixture.Paraffin is used to displace the dissolving compound that has saturatedthe specimens. The impregnation takes an additional 20 minutes.

TABLE 6.0 Composition of Dissolving Compound Component Weight-percentAcetone 59 Hexane 41

TABLE 6.1 Typical processing times and specimen type. ProcessingImpregnation Tissue Reference No. Time (minutes) Time (min) Type 501 1050 Breast tissue 2 mm 502 10 50 liver 2 mm 503 10 50 liver 2 mm 504 2050 liver 5 mm 505 30 50 Breast tissue 5 mm 506 30 50 Breast tissue 5 mm507 20 50 liver 5 mm 508 30 50 Breast tissue 4 mm 509 30 50 liver 10 mm510 30 50 Liver 10 mm 511 30 50 Breast tissue 2 mm 512 30 50 Breasttissue 4 mm

The dissolving compound of Table 6.0 is loaded into the solventregenerator and heated for 15 minutes to an equilibrium temperature of60° C. and a resulting pressure of about 0.8 bars (12 psig), thentransferred to the reaction container 11 holding the specimens listed inTable 10.1.

The reaction container conditions are next equilibrated to 60° C., about0.8 bars (12 psig), a flow rate of 2.35 liter/min, and a space velocityof 91.2-sec. Specimens are processed for individual times as indicatedin Table 10.1. After this processing, specimens are super-saturated withthe compound. The excess compound is then returned to the regenerator.

Paraffin is next charged into the reaction container. The container isstabilized to 60° C., and a chemical-potential difference is createdbetween the paraffin and compound-saturated specimens. Vacuum is thenused to increase this differential, vaporizing the compound within thespecimen, and substituting compound molecules with hot paraffin. Vacuumis continually applied until a pressure equilibration is achieved. Tenminutes after equilibrium is obtained, the heaters are disengaged, thevent solenoid opens, and the reaction container is brought to roomtemperature before extracting the specimens.

Example 7

The following is an example of batch solvent blending within thereaction container 11. Table 7.0 shows wt % of components before andafter batch reactor blending.

TABLE 7.0 Solvent Batch blending Composition of Dissolving compoundbefore and after. Wt % Before Reactor Wt % After Reactor ComponentBlending Blending Acetone 59.0 54.0 Hexane 41.0 38.0 Paraffin — 8.0

TABLE 7.1 Typical processing times and specimen type. ProcessingImpregnation Tissue Reference No. Time (minutes) Time (min) Type 601 1050 Breast tissue 2 mm 602 10 50 liver 2 mm 603 10 50 liver 2 mm 604 2050 liver 5 mm 605 30 50 Breast tissue 5 mm 606 30 50 Breast tissue 5 mm607 20 50 liver 5 mm 608 30 50 Breast tissue 4 mm 609 30 50 liver 10 mm610 30 50 Liver 10 mm 611 30 50 Breast tissue 2 mm 612 30 50 Breasttissue 4 m

The dissolving compound residing in the solvent regenerator isconditioned by heating it to an equilibrium temperature of 60° C. and apressure of about 0.8 bars (12 psig), then transferred to the reactioncontainer 11 holding the tissue specimens. Any excess compound isreturned to the solvent regenerator.

The reaction container conditions are next equilibrated to 60° C., about1.6 bars (25 psig), a flow rate of 2.35 liter/min, and a space velocityof 91.2-sec. Specimens are processed for individual times as indicatedin Table 7.1. After this processing, specimens are super-saturated withthe dissolving compound.

Next, the reaction container is flooded with paraffin. The container isrestabilized to 60° C., and a chemical-potential difference is createdbetween the paraffin and the dissolving compound-saturated specimens.Vacuum is then used to increase this differential, vaporizing thecompound within the specimens. The compound molecules are replaced withhot paraffin. Vacuum is continually applied until a pressureequilibration is achieved. Ten minutes after equilibrium is obtained,the heaters are disengaged, the vent solenoid opens, and the reactioncontainer is brought to room temperature before extracting thespecimens.

The present technique provides consistent, lifelike results, faster,without pre-processing requirement, with less cutting (often, specimensup to 5 mm thick can be processed), without solvent cross-contaminationproblems, without having to burr specimens, without water contaminationproblems, and in a single reaction container without the safety problemsassociated with microwave systems.

The present embodiments do not use microwaves to heat the specimen anddoes not use microwave reagents and thus do not suffer the disadvantageswhich plague microwave processing. Therefore, a homogeneous temperaturethroughout the reaction container can be readily maintained, andhomogeneous temperature profiles can be seen between specimens anddissolving compound.

In the disclosed embodiments the presence of water in the dissolvingcompound does not affect the reactor container's temperature profile orthe specimen's lifelike characteristics as can happen with microwaveprocessing. Excess water from cellular solute extract and other cellularmolecules can be further separated during solvent regeneration.

The solvent extraction process can take place in a single pressurizedreactor container at pressures of between −29.9 inches Hg to 3.3 bars(50 psig), extraction temperatures between 30° C. and 100° C., andtypical space velocities between 30 and 150 seconds.

In some embodiments paraffin need not be separated from the dissolvingcompound in a separate vessel, and there are no robotic or manualtransfers of specimens.

In some embodiments the paraffin concentration in the dissolvingcompound can be allowed to increase over the life of the dissolvingcompound without affecting the lifelike characteristics of thespecimens. In some embodiments the dissolving compound/paraffin blendcan also be used to lubricate pumps used in the processor. In someembodiments the dissolving compound/paraffin blend can be chemicallystable.

For many tissue types and sizes the specimen processing described abovecan be completed in approximately one hour. Specimens as large as 5 mmcan be readily processed. There can be little or no significantpre-processing of specimens prior to dissolving compound extraction.

In some embodiments paraffin impregnation of the specimens does notrequire vacuum drying or transfer to another vessel prior to paraffinimpregnation as with microwave processing. Specimen and dissolvingcompound temperatures can be homogenous within the reaction containeronce equilibrium has been reached.

Other tissue types can be processed using the present inventionembodiments, such as but not limited to appendix, bowel, fallopian tube,kidney, liver, lung, parotid, placenta, prostate, thyroid, adenoma,cervix, skin and many others.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A process for preserving a specimen comprising:removing from the specimen cellular solutes that interfere withpreservation and replacing the removed solutes with an impregnatedsupport medium; wherein the removing comprises extracting an amount ofwater supplied by the specimen; wherein the extracting comprises:dissolving the solutes including the amount of water using a dissolvingcompound; forming an azeotropic water-containing mixture of the amountof water and a component of the dissolving compound; azteotropicallyvaporizing off an amount of the azeotropic water-containing mixture;forming a second azeotropic mixture of at least two components of thedissolving compound; and azteotropically vaporizing off an amount of thesecond azeotropic mixture.
 2. The process of claim 1 wherein theremoving further comprises: clearing an amount of the dissolvingcompound from the specimen; wherein the clearing comprises: forming anazeotropic fixitive-containing mixture of an amount of a fixitive agentcomponent present in the dissolving compound and another component inthe dissolving compound; and azteotropically vaporizing off an amount ofthe azeotropic fixitive-containing mixture.
 3. The process of claim 1wherein the removing further comprises: clearing an amount of thedissolving compound from the tissue; wherein the clearing comprises:forming an azeotropic dehydrant-containing mixture of an amount of adehydrant agent component present in the dissolving compound and anothercomponent in the dissolving compound; and azteotropically vaporizing offan amount of the azeotropic dehydrant-containing mixture.
 4. The processof claim 1 wherein the impregnated support medium comprises paraffin. 5.The process of claim 1 wherein the impregnated support medium isselected from the group consisting of: paraffin, plastic polymers,polyvinyl alcohol, polyethylene glycols, waxes, cellulose derivatives,agars, gelations, and sugars.
 6. The process of claim 1 wherein thedissolving compound comprises a single component or groups of componentsthat is removed by vaporization from the specimen without damage to thespecimen.
 7. The process of claim 1 wherein the dissolving compoundcomprises a single component or groups of components that releaseformaldehyde from the specimen and convert it to a monomeric gas.
 8. Theprocess of claim 1 wherein the dissolving compound further comprises asupport medium component.
 9. The process of claim 1, wherein thedissolving compound comprises a fixative component; a dehydrantcomponent; and a clearing agent component.
 10. The process of claim 9wherein the dissolving compound further comprises a cation as a cellularanti-rupturing agent.
 11. The process of claim 10 wherein the cationcellular anti-rupturing agent comprises a compound selected from thegroup consisting of: zinc acetate, iron acetate, magnesium acetate, zinccitrate, iron citrate, and zinc malate.
 12. The process of claim 9wherein the fixative component is selected from the group consisting of:formalin, glacial acetic acid, zinc chloride or zinc sulfate, chromicacid, ethanol, methanol, acetone, mercuric chloride, picric acid, andpotassium dichromate.
 13. The process of claim 9 wherein the dehydrantcomponent is selected from the group consisting of: alcohols, isopropylalcohol, ethanol, denatured alcohols, butanols, ethers, ketones,dioxane, and acetone.
 14. The process of claim 9 wherein the clearingagent component is selected from the group consisting of: xylene,toluene, benzene, acetone, butanols, dioxane, methyl salicylate,cedarwood oil, hexane, heptane, octane, and decane.
 15. The process ofclaim 9 wherein the dielectric constant of the clearing agent componentis between 2.0 and
 10. 16. The process of claim 1 wherein the dissolvingcompound comprises a mixture of between about 0 and 30% by weightalcohol, between about 45 and 99% by weight ether, and between about 0and 20% by weight hydrocarbon.
 17. The process of claim 1 furthercomprising prefixing the specimen using an amount of fixativecomprising: a mixture of between about 20 and 50% by weight methanol,and between about 20 and 50% by weight ethanol; wherein the mixture issaturated with anhydrous ammonia; wherein the mixture has a pHmaintained between about 5.0 and 5.4 through the application of aneffective amount of glacial acetic acid.
 18. The process of claim 17further comprising the mixture including: an amount ofvanadyl-ribonucleoside complex or RNAlater® as a nucleic acidpreservative.
 19. The process of claim 1, further comprising prefixingthe specimen using an amount of fixative comprising: a mixture ofbetween about 80 and 98% by weight methanol, between about 1 and 20% byweight acetonitrile, and between about 1 and 5% by weight EDTA; whereinthe mixture has a pH maintained between about 5.0 and 5.4 through theapplication of an effective amount of glacial acetic acid.